CN116622538A - A Strain of Vibrio Natrium and Its Application - Google Patents

Abstract

The invention relates to the technical field of microbes, in particular to a strain of vibrio natrium and its application. The present invention isolates a strain of Vibrio natriegens V‑Z9 from the large seaweed Gracilaria, which was preserved in the China Center for Type Culture Collection on March 23, 2022, with the preservation number CGMCC No.24570. Compared with the existing sodium-demanding Vibrio, the strain can fully degrade λ-carrageenan into λ-carrageenan oligosaccharides with a molecular weight of 10-30 kDa, and the obtained λ-carrageenan oligosaccharides have significant antioxidant effects.

Description

A Sodium Vibrio Strain and Its Application

technical field

The invention relates to the technical field of microbiology, in particular to a strain of vibrio natrium and its application.

Background technique

Large seaweed is a valuable resource for developing marine polysaccharide functional foods and finding new polysaccharide drug lead compounds. It contains a variety of λ-carrageenan polysaccharides with different structures. At present, there is a large gap in the supply of λ-carrageenan polysaccharide resources, and the production methods are backward. However, λ-carrageenan polysaccharides have problems such as large molecular weight, low water solubility, and difficult absorption. Therefore, λ-carrageenan polysaccharides are only sold abroad as raw materials or simply adopted Extensive processing to extract λ-carrageenan as food or food additives.

Compared with λ-carrageenan polysaccharides, λ-carrageenan oligosaccharides not only have rich sources, various types, stable properties, wide application, and unique activity, but also have significant advantages such as small molecular weight, easy solubility in water, and no antigenicity. A variety of medical and health effects, such as anti-virus, anti-oxidation, anti-coagulation, immune regulation and other functions.

Common methods for preparing λ-carrageenan oligosaccharides mainly include chemical and physical methods, but existing methods have disadvantages such as destroying the structure of polysaccharides and producing harmful by-products.

Contents of the invention

The invention provides a strain of natrivibrio and its application to overcome at least one technical problem in the prior art.

In view of this, the present invention provides a strain of vibrio natrium and application thereof, the bacterial strain can degrade λ-carrageenan into λ-carrageenan oligosaccharide, the degradation rate is greater than 85%, and the obtained λ-carrageenan oligosaccharide has Significant antioxidant effect.

The present invention isolates and obtains the bacterial strain V-Z9 from the large seaweed Gracilaria, the bacterial strain is a gram-negative bacillus, the colony is moist, light yellow, round, with neat edges, aerobic bacteria, and can degrade λ-carrageenan and cultured on a solid There are hydrolysis circles on the base, and there are obvious transparent circles after staining with Lushi's iodine solution. The nucleotide sequence of the 16S rRNA gene of the strain is shown in SEQ ID NO:1. It is identified as Vibrio natriegens by morphology and 16srRNA gene, and its deposit number is CGMCCNo.24570.

The present invention also provides the application of the sodium-demanding Vibrio in the preparation of λ-carrageenan oligosaccharides.

The present invention also provides a method for preparing λ-carrageenan oligosaccharides, which is prepared by degrading λ-carrageenan by the Vibrio nadivorum V-Z9 described in the present invention.

In some embodiments, the method for preparing λ-carrageenan oligosaccharides provided by the invention specifically includes:

Step (1): inoculating the Nadidemanding Vibrio with the preservation number CGMCC No.24570 into the seed medium, culturing, and obtaining the seed liquid;

Step (2): inoculating the seed liquid in a liquid medium containing λ-carrageenan, culturing, centrifuging to remove the bacteria, and obtaining a supernatant;

Step (3): subjecting the supernatant to molecular interception and centrifugation to collect components with a molecular weight of 10-30 kDa.

In some embodiments, the inoculum amount of Narophilus V-Z9 described in step (1) is 2-3% (v/v); in some specific embodiments, the inoculum amount can be specifically 3% (v /v).

In some embodiments, the culture conditions are as follows: the rotation speed is 160-180 rpm, at 25-30°C, for 12-15 hours; in some specific examples, the culture conditions are: the rotation speed is 180 rpm, at 30°C, Cultivate for 15h.

In some specific embodiments, in step (2), the liquid medium containing λ-carrageenan includes the following components in mass and volume percentages:

0.15% λ-carrageenan, 2.5% NaCl, 0.35% (NH ₄ ) ₂ SO ₄ , 0.1% K ₂ HPO ₄ 3H ₂ O, 0.2% MgSO ₄ 7H ₂ O, 0.02% FeSO ₄ 7H ₂ O, The pH is 7.

In some embodiments, the inoculum amount of the seed solution in step (2) is 1-3% (v/v); in some specific examples, the inoculum amount can be specifically 3% (v/v).

In some embodiments, the cultivation conditions in step (2) are as follows: the rotation speed is 160-180 rpm, at 25-30°C for 50-60 hours; in some specific examples, the cultivation conditions are: the rotation speed is 180 rpm , at 30°C for 50h.

The present invention also provides λ-carrageenan oligosaccharides prepared by the method of the present invention.

The present invention has studied the antioxidant property of the λ-carrageenan oligosaccharide (the thick λ-carrageenan oligosaccharide of molecular weight 10～30kDa) that is made by the method described in the present invention, finds its DPPH scavenging rate at pH 6.5～7.5 The DPPH scavenging rate at a temperature of 25-30° C. is >85%, and has a significant antioxidant effect.

Based on the above effects, the present invention also provides the application of the λ-carrageenan oligosaccharide in the preparation of antioxidant products.

The present invention isolates a strain of Vibrio natriegens V-Z9 from the large seaweed Gracilaria, and preserved it in the China Center for Type Culture Collection on March 23, 2022, with the preservation number CGMCCNo.24570. The strain can fully degrade λ-carrageenan into λ-carrageenan oligosaccharides with a molecular weight of 10-30 kDa, and the obtained λ-carrageenan oligosaccharides have significant antioxidant effects.

Biological Deposit Instructions

V-Z9, taxonomic designation: Vibrio natriegens, preserved on March 23, 2022, in the General Microbiology Center of China Committee for the Collection of Microbial Cultures, the address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, the deposit number is CGMCCNo.24570.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. Those skilled in the art can also obtain other drawings based on these drawings without creative work.

Fig. 1 is strain morphological diagram;

Figure 2 is a scanning electron microscope image of the bacterial strain;

Fig. 3 is the hydrolysis circle (Lushi's iodine solution) figure of bacterial strain degradation lambda-carrageenan;

Figure 4 is a phylogenetic tree diagram of strain V-Z9;

Fig. 5 is the antioxidant activity of λ-carrageenan oligosaccharide of 10～30kDa;

Fig. 6 is the influence of temperature on the oxidation resistance of λ-carrageenan oligosaccharide;

Figure 7 is the effect of pH on the antioxidant activity of λ-carrageenan oligosaccharides;

Fig. 8 is the antioxidant activity of 10-30 kDa λ-carrageenan oligosaccharides in the fermentation broth at different time periods.

Detailed ways

The invention discloses a strain of vibrio natriurum and its application. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.

Below in conjunction with embodiment, further set forth the present invention:

Example 1 Screening and Identification of Vibrio natriegens V-Z9

1.1 Medium

(1) The mass volume percentage of each component in the λ-carrageenan liquid medium is: 0.15% λ-carrageenan, 2.5% NaCl, 0.35% (NH ₄ ) ₂ SO ₄ , 0.1% K ₂ HPO ₄ · 3H ₂ O, 0.2% MgSO ₄ ·7H ₂ O, 0.02% FeSO ₄ ·7H ₂ O, pH 7.

(2) The mass volume percentage of each component in the λ-carrageenan solid medium is: 1.2% agar, 2% λ-carrageenan, 2.5% NaCl, 0.35% (NH ₄ ) ₂ SO ₄ , 0.1% K ₂ HPO ₄ .3H ₂ O, 0.2% MgSO ₄ .7H ₂ O, 0.02% FeSO ₄ .7H ₂ O, pH 7.

1.2 Strain screening

1.2.1 Sample collection: Collect the large seaweed Gracilaria off the coast of Hainan, take 5g sample, add 50mL PBS buffer, grind it in a sterile grinder, pour it into a 100mL EP tube, shake it up and down, and let it stand for 10 minutes before use.

1.2.2 Preliminary screening of strain V-Z9: Take 10 μL of processed Gracilaria samples and inoculate them into a liquid medium with λ-carrageenan as the sole carbon source for fermentation, culture at 30°C and 180rpm for 48 hours, and 3 passages.

1.2.3 Re-screening of strain V-Z9: Take 3 μL of subcultured fermentation broth and spread it on the λ-carrageenan solid medium, and after the colonies grow, perform multiple streaks until a single colony is isolated (Figure 1) .

1.3 Morphological characteristics of strain V-Z9

Strain V-Z9 is a gram-negative bacterium, and it is a bacillus. The colony is moist, light yellow, round, with neat edges, an aerobic bacterium, and can degrade λ-carrageenan and has a hydrolysis circle on the solid medium (Figure 1- 2), and after staining with Lugol's iodine solution, there is an obvious transparent circle (stained with 5% Lugol's iodine solution) (Figure 3).

1.4 Molecular biological identification of strain V-Z9

After the genome of V-Z9 was extracted, 16S rDNA sequencing and identification were performed. The strain was identified as Vibrio natriegens, and we named it Vibrio natriegens V-Z9 (Fig. 4).

The whole 16S rRNA gene sequence is 1374bp long, and the sequence is shown in SEQ ID NO:1.

1.5 Method for preparing λ-carrageenan oligosaccharides from strain V-Z9

(1) Inoculate the 2216E seed culture medium with a 3% (v/v) inoculum amount of Narophilus V-Z9, rotate at 180 rpm, fill with a liquid volume of 30%, and incubate at 30° C. for 15 hours to obtain a seed liquid.

(2) Inoculate the seed solution in λ-carrageenan liquid medium with an inoculation amount of 3% (the medium for preparing λ-carrageenan oligosaccharides is λ-carrageenan liquid medium: 0.15% λ-carrageenan, 2.5 %NaCl, 0.35% (NH4) 2SO4, 0.1% K2HPO4 3H2O, 0.2% MgSO4 7H2O, 0.02% FeSO4 7H2O, pH 7). 180rpm, culture at 30°C for 60h, obtain the fermentation broth, remove the bacterial cells by centrifugation, and obtain the supernatant;

(3) The supernatant was obtained by the method of molecular interception, using an Ultra centrifuge tube to obtain crude λ-carrageenan oligosaccharides with a molecular weight of 10-30 kDa.

1.6 Antioxidant characteristics of V-Z9λ-carrageenan oligosaccharides

1.6.1 Antioxidant activity of 10～30kDaλ-carrageenan oligosaccharides

The antioxidant properties of λ-carrageenan oligosaccharides were evaluated using the DPPH radical scavenging method. Prepare a 0.1 mM DPPH solution using absolute ethanol. Firstly, the antioxidant properties of 10-30kDa λ-carrageenan oligosaccharides were investigated. Mix 50 μL DPPH solution with 150 μL 10-30 kDa λ-carrageenan oligosaccharides (pH 7.0), and incubate at 25°C for 60 minutes in the dark. , Measure the absorbance of the reaction solution at 517nm, and replace the sample with absolute ethanol as a negative control, and calculate the antioxidant efficiency of λ-carrageenan oligosaccharides of 10-30kDa. It was found that the DPPH clearance rate of λ-carrageenan oligosaccharides of 10 to 30 kDa after molecular interception was 85%, which was nearly 57% higher than that of the same volume of fermentation broth in step (2) of Section 1.5, showing a strong Antioxidant properties (Figure 5).

1.6.2 Effect of temperature on the antioxidant activity of λ-carrageenan oligosaccharides

Mix 50 μL DPPH solution with 150 μL 10-30 kDa λ-carrageenan oligosaccharide solution (pH 7.0), incubate at different temperatures for 60 min in the dark, measure the absorbance of the reaction solution at 517 nm, and replace the sample with absolute ethanol as As a negative control, the antioxidant efficiency of λ-carrageenan oligosaccharides of 10 to 30 kDa was investigated at different temperatures. The results showed that the DPPH clearance rate in the temperature range of 25 to 35 ° C was the best and both > 85% (Fig. 6-7 ).

1.6.3 The effect of pH on the antioxidant activity of λ-carrageenan oligosaccharides

First, adjust the pH of the 10-30 kDa λ-carrageenan oligosaccharide solution to 5-11. Then, mix 50 μL of LDPPH solution with 150 μL of 10-30 kDa λ-carrageenan oligosaccharide solution, incubate at 30°C in the dark for 60 minutes, measure the absorbance of the reaction solution at 517 nm, and replace the sample with absolute ethanol as a negative control , to investigate the antioxidant efficiency of 10-30kDa λ-carrageenan oligosaccharides at different temperatures, the results showed that the DPPH clearance rate in the pH range of 6-7.5 was the best and all > 85%.

1.6.4 Antioxidant activity of 10-30kDa λ-carrageenan oligosaccharides in fermentation broth at different time periods

Fermentation liquids in different fermentation stages were selected, centrifuged to remove bacterial cells, and supernatant liquid was obtained. The supernatant is passed through the method of molecular interception to obtain crude lambda-carrageenan oligosaccharides with a molecular weight of 10-30 kDa at different fermentation stages. Mix 50 μL of DPPH solution with 150 μL of 10-30 kDa λ-carrageenan oligosaccharide solution (pH 7.0) at various time intervals (pH 7.0), incubate at 30°C in the dark for 60 min, measure the absorbance of the reaction solution at 517 nm, and measure the absorbance of the reaction solution in anhydrous Ethanol was used as a negative control instead of samples to investigate the antioxidant efficiency of 10-30kDaλ-carrageenan oligosaccharides in the fermentation broth at different time periods. The results showed that the DPPH clearance rate of 10-30kDaKλ-carrageenan oligosaccharides in the fermentation broth after It reaches the optimum of about 85%, and then enters a plateau (60-90 h). After the plateau, the clearance rate of 10-30 kDa λ-carrageenan oligosaccharides in the fermentation broth to DPPH gradually decreases ( FIG. 8 ).

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (10)

1. Vibrio natrii (Vibrio natriegens) with preservation number of CGMCC No.24570.

2. The vibrio natrii of claim 1, wherein the nucleotide sequence of the 16S rRNA gene is set forth in SEQ ID NO: 1.

3. Use of vibrio natrii in accordance with claim 1 or 2 for preparing lambda-carrageenan oligosaccharides.

4. A process for the preparation of lambda-carrageenan oligosaccharides, characterized in that it is prepared by degrading lambda-carrageenan by vibrio natrii according to claim 1 or 2.

5. The method according to claim 4, comprising:

step (1): inoculating vibrio natrii with the preservation number of CGMCC No.24570 into a seed culture medium, and culturing to obtain seed liquid;

step (2): inoculating the seed liquid into a liquid culture medium containing lambda-carrageenan, culturing, centrifuging to remove thalli, and obtaining supernatant;

step (3): and (3) carrying out molecular interception and centrifugation on the supernatant, and collecting components with the molecular weight of 10-30 kDa.

6. The method according to claim 5, wherein in the step (1), the inoculating amount of the Vibrio natrii V-Z9 is 2-3% (V/V); the culture conditions are as follows: the rotation speed is 160-180 rpm, and the culture is carried out for 12-15 h at 25-30 ℃.

7. The method according to claim 5, wherein in the step (2), the mass and volume percentages of each component in the liquid medium containing lambda-carrageenan are as follows:

0.15% lambda carrageenan, 2.5% NaCl, 0.35% (NH) ₄ ) ₂ SO4、0.1％K ₂ HPO ₄ ·3H ₂ O、0.2％MgSO ₄ ·7H ² O、0.02％FeSO ₄ ·7H ₂ O, pH 7.

8. The method according to claim 5, wherein in the step (2), the seed liquid is inoculated in an amount of 1 to 3% (v/v); the culture conditions are as follows: the rotation speed is 160-180 rpm, and the culture is carried out for 50-60 h at the temperature of 25-30 ℃.

9. Lambda-carrageenan oligosaccharides obtainable by the process according to any of claims 4 to 8.

10. Use of a lambda-carrageenan oligosaccharide according to claim 9 for the preparation of an antioxidant product.